The present invention relates to a process for dissolving oxygen in an aqueous liquor, a process for culturing cells in an oxygen-containing aqueous liquor obtained by the oxygen-dissolving process, and apparatus for practicing these processes.
In the present specification, the word "cells" includes animal cells, plant cells and microorganism cells unless otherwise specified.
The cultivation of cells, in particular, animal cells, is an important technique for producing useful materials such as drugs. In the aerobic cultivation of animal cells or microorganism cells, it is often necessary to supply oxygen dissolved in an aqueous liquor in an amount sufficient to meet the demand for oxygen the consumption of which increases with an increase of the cell concentration.
As methods for supplying oxygen, there have been known the following methods.
1) A method of blowing oxygen directly through a liquor.
2) A method of blowing an oxygen-containing gas against the free surface of a culture liquor and thereby diffusing oxygen from the surface of the liquor to the inside to dissolve oxygen in the liquor.
3) A method of placing an oxygen-permeable solid membrane so as to bring one side into contact with an oxygen-containing gas and the other side with a liquor, and thereby allowing oxygen to permeate the solid membrane and diffuse into the liquor (Development of Biological Standard, Vol. 66, p. 263-268).
4) A method similar to the method 3), in which the pores of a porous hollow-fiber membrane are plugged with an oxygen-permeable solid material, and oxygen which has passed through the hollow-fiber membrane is diffused into and dissolved in a liquor (see Jap. Pat. Appln. Kokai (Laid-Open) No. 61-18402).
On the other hand, animal cells are roughly classified to anchorage-independent cells and anchorage-dependent cells according as their propagation requires or does not require a solid surface. For the propagation of the anchorage-independent cells, the method 1) of blowing oxygen directly through a liquor is suitable. For the propagation of the anchorage-dependent cells, a culturing method in which animal cells are attached to micro-beads is often employed, and therefore a suitable oxygen-supplying method is the method 2) of blowing an oxygen-containing gas against the free surface of a culture liquor, or the method 3) or 4) which use an oxygen-permeable solid membrane or hollow-fiber membrane, respectively. The methods using the solid membrane or hollow-fiber membrane permit more efficient oxygen supply than does the method of blowing an oxygen-containing gas against the free surface of a culture liquor, but the amount of oxygen which can be supplied by the methods is limited, and when the methods are employed for highly productive high-density cell-culturing, the amount of oxygen supplied tends to be insufficient.
In particular, the method disclosed in Jap. Pat. Appln. Kokai (Laid-Open) No. 61-18402 does not always permit sufficient supply of oxygen because the material used for the plugging becomes a solid after molding.
In addition, fluorocarbons (organofluorine compounds) are known as solvents having a high affinity for oxygen. They have been noted as materials for artificial blood for a long time and have already been used in a certain type of artificial lung. There are known cases where the principle of the artificial lung is used as it is in an oxygen-supplying means for culturing animal cells (see Jap. Pat. Appln. Kokai (Laid-Open) Nos. 61-1383 and 64-60368).
In these cases, a volatile fluorocarbon and oxygen are brought into contact with each other outside a culture liquor tank, whereby oxygen is supported on the fluorocarbon. The fluorocarbon thus treated is added dropwise to a culture liquor in the culture liquor tank, and oxygen is diffused into the liquor during the precipitation of the fluorocarbon. Since the fluorocarbon having a high specific gravity precipitates finally in the bottom of the tank to undergo phase separation between it and the culture liquor, the fluorocarbon in the tank bottom is recovered by a suitable means, taken out of the tank, and then brought into contact with oxygen again to re-support oxygen.
However, in the method described above, the fluorocarbon which is expensive is used in a large amount and must be recycled continuously for a long period of time (1 to 2 months). Moreover, a considerable amount of the fluorocarbon is lost due to its volatility for this period. Therefore, the method does not meet the real demand for a low-cost, simple oxygen-supplying method.